{"gene":"TNC","run_date":"2026-04-28T21:42:59","timeline":{"discoveries":[{"year":1992,"finding":"The crystal structure of a fibronectin type III (FNIII) domain from tenascin-C was solved by multiwavelength anomalous diffraction (MAD) phasing of the selenomethionyl protein to 1.8 Å resolution, revealing a folding topology identical to extracellular domains of the human growth hormone receptor and CD4, and distinct from but related to immunoglobulin constant domains. An RGD sequence mediating cell adhesion was found in a tight turn on an exposed loop.","method":"X-ray crystallography (MAD phasing, selenomethionyl protein), 1.8 Å resolution","journal":"Science","confidence":"High","confidence_rationale":"Tier 1 — crystal structure with full atomic resolution, foundational structural paper","pmids":["1279805"],"is_preprint":false},{"year":1991,"finding":"The complete primary structure of human tenascin-C was established by cDNA sequencing, identifying 14.5 EGF-like repeats and 15 fibronectin type III repeats (versus 11 in chicken). At least four different isoforms containing different numbers of FNIII repeats were identified by PCR, demonstrating alternative splicing of the TNC pre-mRNA. Monoclonal antibody BC-2 was mapped to an alternatively spliced FNIII repeat, while BC-4 recognized an EGF-like epitope present in all isoforms.","method":"cDNA sequencing, PCR amplification of splice variants, expression vector library screening, epitope mapping","journal":"Nucleic Acids Research","confidence":"High","confidence_rationale":"Tier 1 — primary structure determination with functional domain mapping and splice variant identification","pmids":["1707164"],"is_preprint":false},{"year":1993,"finding":"Multiple integrins of the αv subtype mediate cell attachment to the third FNIII repeat of tenascin-C (cytotactin) containing the RGD tripeptide. However, mutation of RGD to RAD did not abolish attachment, and anti-β1 antibodies partially inhibited binding to intact TNC but not the FNIII repeat, indicating additional integrin receptors. Thus TNC engages multiple integrin receptor subtypes through partially RGD-dependent and -independent mechanisms.","method":"Recombinant fusion protein cell attachment assays, RGD peptide competition, anti-integrin blocking antibodies","journal":"Proceedings of the National Academy of Sciences","confidence":"High","confidence_rationale":"Tier 2 — reciprocal blocking antibody experiments with recombinant domain fragments, replicated across cell lines","pmids":["7694284"],"is_preprint":false},{"year":1993,"finding":"Human umbilical vein endothelial cells attach to and spread on human tenascin-C via two distinct integrin receptors: α2β1 mediates cell spreading (inhibited by anti-α2 and anti-β1 antibodies), and αvβ3 mediates partial cell attachment via the SRRGDMS site (inhibited by LM609). Both integrins were affinity-purified on tenascin-C columns. The α2β1 binding site on TNC is distinct from the RGD site.","method":"Anti-integrin blocking antibodies, affinity chromatography on TNC-coated columns, radio-receptor binding assay","journal":"Journal of Cell Science","confidence":"High","confidence_rationale":"Tier 2 — affinity purification combined with blocking antibodies, identifying two distinct receptor-ligand interactions","pmids":["7693733"],"is_preprint":false},{"year":1994,"finding":"The alternatively spliced segment of tenascin-C (TNfnA-D) binds specifically to annexin II on the cell surface of glioma and endothelial cells with ~2 nM affinity (~2–5 × 10^5 binding sites/cell). Annexin II was identified by Scatchard analysis, blot binding assays, affinity chromatography, and protein sequencing. Anti-annexin II antibodies blocked TNfnA-D binding to live cells, establishing annexin II as a cell-surface receptor for the alternatively spliced region of TNC.","method":"Radioligand binding assay, Scatchard analysis, affinity chromatography, protein sequencing, blocking antibodies","journal":"Journal of Cell Biology","confidence":"High","confidence_rationale":"Tier 1–2 — multiple orthogonal methods including Scatchard binding kinetics, affinity purification, and protein sequencing","pmids":["7518469"],"is_preprint":false},{"year":1994,"finding":"The integrin α9β1 mediates cell attachment to the third FNIII repeat of tenascin-C at a site that is not the RGD tripeptide: RGD peptide competition and RGD→RAD/RAA mutations did not inhibit α9β1 binding. α9β1 did not mediate attachment to fibronectin, laminin, vitronectin, fibrinogen, thrombospondin, or collagens, identifying tenascin-C as a specific ligand for this integrin.","method":"Stable transfection of α9 cDNA in 293 and SW480 cells, cell adhesion assays with recombinant TNC fragments, RGD peptide competition, mutagenesis of RGD site","journal":"Journal of Biological Chemistry","confidence":"High","confidence_rationale":"Tier 1–2 — transfection + recombinant fragment mapping + mutagenesis establishing a non-RGD binding mechanism","pmids":["7523411"],"is_preprint":false},{"year":1995,"finding":"The integrin α8β1 functions as a receptor for tenascin-C, fibronectin, and vitronectin. α8β1 binds to the RGD-containing third FNIII repeat of tenascin-C (eluted by GRGDSP peptide), localizes to focal contacts on fibronectin and vitronectin substrata, and mediates adhesion of α8-transfected 293 cells. Among RGD-containing ECM proteins, only tenascin was able to mediate adhesion via α8β1 in addition to fibronectin and vitronectin.","method":"Stable transfection of α8 cDNA, cell adhesion assays, affinity chromatography on fibronectin/vitronectin-Sepharose with RGD peptide elution, recombinant TNC fragment binding","journal":"Journal of Biological Chemistry","confidence":"High","confidence_rationale":"Tier 1–2 — transfection + affinity chromatography + recombinant fragment assays","pmids":["7559467"],"is_preprint":false},{"year":1997,"finding":"Tenascin-C promotes survival and EGF-dependent growth of vascular smooth muscle cells (SMCs) through interaction with αvβ3 integrins. TNC-αvβ3 interaction redistributes filamentous actin to focal adhesion complexes that colocalize with EGF receptor clusters, increasing EGF-R tyrosine phosphorylation and activation. Cross-linking β3 integrins replicates TNC effects on EGF-R clustering and phosphorylation. MMP inhibition (GM6001) suppresses TNC expression; denatured collagen induces TNC in a β3-integrin-dependent manner.","method":"Exogenous TNC addition to SMCs, MMP inhibitor treatment, floating collagen gel assays, β3-integrin cross-linking, EGF-R phosphorylation assays, actin/EGF-R colocalization by immunofluorescence","journal":"Journal of Cell Biology","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal experiments linking TNC-αvβ3 interaction to EGF-R clustering and phosphorylation with pharmacological and antibody interventions","pmids":["9314546"],"is_preprint":false},{"year":2001,"finding":"Select EGF-like repeats of tenascin-C directly bind to and activate the EGF receptor (EGFR), inducing EGFR autophosphorylation and ERK/MAPK activation, and stimulating mitogenesis. Micromolar concentrations of EGF-like repeats induced EGFR autophosphorylation to levels comparable to subsaturating EGF. When tethered to inert beads (simulating hexabrachion presentation), EGF-like repeats mediated EGFR-dependent adhesion. Specific EGFR binding was confirmed by immunofluorescence and EGFR cross-linking, both abolished by EGF competition.","method":"EGFR autophosphorylation assays, ERK/MAPK activation, mitogenesis assay, bead-mediated adhesion, immunofluorescence, EGF competition, EGFR cross-linking","journal":"Journal of Cell Biology","confidence":"High","confidence_rationale":"Tier 1–2 — multiple biochemical assays plus competition experiments establishing TNC EGF-like repeats as direct EGFR ligands","pmids":["11470832"],"is_preprint":false},{"year":2001,"finding":"Tenascin-C directly binds to the 13th fibronectin type III repeat (FNIII13) of fibronectin, blocking syndecan-4 binding to fibronectin's HepII site. This inhibits integrin signaling (syndecan-4 coreceptor function) and disrupts cell adhesion while promoting tumor cell proliferation. Overexpression of syndecan-4 (but not syndecan-1 or -2) rescued the adhesion defect, and addition of FNIII13 restored adhesion and normalized proliferation.","method":"Mixed fibronectin/TNC substratum adhesion assays, syndecan overexpression (rescue experiment), recombinant FNIII13 competition, cell proliferation assays","journal":"Cancer Research","confidence":"High","confidence_rationale":"Tier 2 — domain-specific binding mapped with recombinant fragments, validated by specific syndecan-4 rescue experiment","pmids":["11731446"],"is_preprint":false},{"year":2004,"finding":"Tenascin-C secreted by myofibroblasts promotes invasion of colon cancer cells by inactivating RhoA via the EGF-like repeats of TNC through EGFR signaling, creating a permissive signal for SF/HGF-activated Rac via c-Met. Myofibroblast-stimulated invasion is characterized by a morphological shift from round (high RhoA, low Rac) to elongated (low RhoA, high Rac) phenotype. Dominant-negative and constitutively active mutants of RhoA-ROCK and Rac, plus pharmacological modulators, confirmed this epistatic pathway: TNC acts upstream of RhoA inactivation, which is required for SF/HGF-Rac-driven invasion.","method":"In vitro invasion assay into collagen I/Matrigel, dominant-negative and constitutively active Rho/Rac mutants, pharmacological inhibitors of RhoA-ROCK and Rac, morphotype analysis, co-culture of myofibroblasts and cancer cells","journal":"FASEB Journal","confidence":"High","confidence_rationale":"Tier 2 — genetic epistasis via dominant-negative/constitutively active mutants plus pharmacological validation, identifying TNC's pathway position","pmids":["15059978"],"is_preprint":false},{"year":2009,"finding":"Tenascin-C is an endogenous activator of Toll-like receptor 4 (TLR4) that drives persistent synovial inflammation. Tnc-knockout mice show rapid resolution of acute joint inflammation and are protected from erosive arthritis. Intra-articular injection of TNC promotes joint inflammation in vivo. In human synovial macrophages and fibroblasts, exogenous TNC induces proinflammatory cytokine synthesis specifically via TLR4 activation. TNC's fibrinogen-like domain was identified as the TLR4-activating region.","method":"Tnc knockout mice (in vivo arthritis model), intra-articular TNC injection, explant cultures from RA synovia, human macrophage/fibroblast stimulation with TNC, TLR4-blocking antibodies and TLR4-deficient cells","journal":"Nature Medicine","confidence":"High","confidence_rationale":"Tier 2 — KO mouse in vivo model + human primary cell experiments + TLR4-blocking validation, replicated across multiple experimental systems","pmids":["19561617"],"is_preprint":false},{"year":2009,"finding":"Periostin promotes incorporation of tenascin-C into the extracellular matrix by acting as a molecular bridge: periostin possesses adjacent domains that bind to TNC and to fibronectin/type I collagen, thereby tethering TNC hexabrachions to ECM fibrils and stabilizing bifurcations to form a meshwork architecture. Both periostin-null and TNC-null mice exhibit a similar phenotype of confined tibial periostitis, and periostin deposition of TNC was demonstrated by in vitro binding assays.","method":"Periostin-null and TNC-null mouse phenotyping, in vitro protein-protein binding assays mapping periostin domains to TNC and fibronectin/collagen","journal":"Journal of Biological Chemistry","confidence":"High","confidence_rationale":"Tier 2 — domain mapping by direct binding assays combined with parallel KO mouse phenotypes showing functional convergence","pmids":["19887451"],"is_preprint":false},{"year":2011,"finding":"TNC promoter activity is dynamically regulated during the cell cycle: live cell imaging of an NIH 3T3 line stably transfected with a TNC promoter-destabilized GFP reporter showed that TNC promoter activity increases during the last 40% of the cell cycle, and that the magnitude of this increase is proportional to promoter activity earlier in the cycle.","method":"Live cell phase-contrast and fluorescence microscopy, destabilized GFP reporter under TNC promoter, automated single-cell tracking over 62 h","journal":"Cytometry Part A","confidence":"Medium","confidence_rationale":"Tier 2 — direct live-cell reporter assay with single-cell resolution, but single lab, single method","pmids":["22045641"],"is_preprint":false},{"year":2013,"finding":"Missense mutations in TNC (c.5317G>A, p.V1773M and c.5368A>T, p.T1796S), located in the conserved fibrinogen-like domain, co-segregate with autosomal dominant progressive nonsyndromic hearing loss (DFNA56) in two Chinese families. TNC is expressed in the basilar membrane and osseous spiral lamina of the cochlea, implicating it in cochlear structural integrity.","method":"Genome-wide linkage analysis, whole exome sequencing, Sanger sequencing validation, mass spectrometry genotyping, segregation analysis in 53 family members","journal":"PLoS One","confidence":"Medium","confidence_rationale":"Tier 3 — genetic segregation with predicted functional effect; no direct functional validation of mutant protein","pmids":["23936043"],"is_preprint":false},{"year":2014,"finding":"Mechanical stress upregulates expression of the alternatively spliced tenascin-C FNIIIA1 domain in osteosarcoma MG-63 cells through the mTOR/4E-BP1/S6K1 signaling pathway. Pharmacological inhibition of mTOR suppressed mechanical stress-induced A1 upregulation, and knockdown of 4E-BP1 and S6K1 reduced basal A1 expression. FNIIIA1 expression promoted MG-63 cell migration.","method":"Cyclic mechanical stress application, mTOR inhibitor treatment, siRNA knockdown of 4E-BP1 and S6K1, RT-PCR and Western blot, cell migration assay","journal":"Molecules and Cells","confidence":"Medium","confidence_rationale":"Tier 2–3 — pharmacological inhibition + siRNA knockdown linking mTOR pathway to mechanically regulated TNC splicing variant expression","pmids":["24598996"],"is_preprint":false},{"year":2016,"finding":"Tenascin-C drives persistence of organ fibrosis by activating TLR4 signaling in fibroblasts. Exogenous TNC stimulates collagen gene expression and myofibroblast transformation via TLR4. Tenascin-C null mice show attenuation of both skin and lung fibrosis (bleomycin model) and accelerated fibrosis resolution. This identifies TNC as an endogenous danger signal that maintains a TLR4-dependent fibrosis amplification loop.","method":"Tenascin-C KO mice (skin and lung fibrosis models), exogenous TNC stimulation of fibroblasts, TLR4 signaling assays, collagen gene expression, myofibroblast transformation assays","journal":"Nature Communications","confidence":"High","confidence_rationale":"Tier 2 — KO mouse models in two fibrosis contexts plus human cell mechanistic experiments, replicating and extending TLR4 mechanism from 2009 arthritis study","pmids":["27256716"],"is_preprint":false},{"year":2016,"finding":"In glioblastoma, tissue stiffness and TNC expression form a positive feedback loop with IDH1 and HIF1α: mutant IDH1 restricts glioma aggression by reducing HIF1α-dependent TNC expression, decreasing ECM stiffness and mechanosignalling. HIF1α directly drives TNC expression; recurrent IDH1-mutant tumors bypass this suppression via reduced miR-203-mediated suppression of HIF1α and TNC. Gain- and loss-of-function xenograft manipulations confirmed that IDH1 mutation reduces TNC-ECM stiffness and tumor aggression.","method":"IDH1 gain- and loss-of-function xenograft models, atomic force microscopy (ECM stiffness), HIF1α manipulation, miR-203 functional studies, patient glioma sample analysis","journal":"Nature Cell Biology","confidence":"High","confidence_rationale":"Tier 2 — in vivo xenograft gain/loss-of-function with AFM mechanical measurements and miRNA mechanistic studies","pmids":["27820599"],"is_preprint":false},{"year":2008,"finding":"EMMPRIN (CD147) overexpression in oral squamous cell carcinoma (SCC) cells promotes tenascin-C deposition in co-cultures with peritumoral fibroblasts, in an MMP-dependent manner (suppressed by the broad-spectrum MMP inhibitor GM6001). EMMPRIN knockdown by siRNA reduced both cell migration and TNC deposition, while EMMPRIN overexpression increased MMP-2, -3, -9 expression and TNC deposition several-fold, establishing an EMMPRIN→MMP→TNC deposition axis.","method":"EMMPRIN retroviral overexpression, siRNA knockdown, co-culture with fibroblasts, MMP inhibitor (GM6001) treatment, TNC deposition quantification, cell migration assay","journal":"Anticancer Research","confidence":"Medium","confidence_rationale":"Tier 3 — gain and loss of function with MMP inhibitor validation in single study","pmids":["18751374"],"is_preprint":false},{"year":2019,"finding":"NG2 proteoglycan binds to tenascin-C via its core protein (chondroitinase treatment did not reduce binding), in a concentration-dependent and saturable manner demonstrated by solid-phase binding assay. Decorin did not bind tenascin-C and could not inhibit NG2-tenascin binding, indicating NG2 uses a distinct domain for tenascin that differs from its collagen-binding region.","method":"Solid-phase binding assay with purified NG2, chondroitinase treatment, decorin competition assay","journal":"Journal of Biological Chemistry","confidence":"Medium","confidence_rationale":"Tier 3 — direct binding assay with competition controls, single study","pmids":["8824254"],"is_preprint":false},{"year":2020,"finding":"Novel TNC-USP6 gene fusions were identified in two cases of primary aneurysmal bone cyst (ABC) by next-generation sequencing, confirmed by RT-PCR and Sanger sequencing. TNC serves as a novel fusion partner driving USP6 rearrangement in this neoplasm.","method":"Next-generation sequencing (NGS), RT-PCR, Sanger sequencing","journal":"Genes, Chromosomes & Cancer","confidence":"Medium","confidence_rationale":"Tier 2 — NGS plus orthogonal RT-PCR/Sanger confirmation; mechanistic implication is structural (fusion driving oncogenesis), but functional consequences not directly tested","pmids":["32011035"],"is_preprint":false},{"year":2021,"finding":"In diabetic kidney disease, TNC activates the TLR4/NF-κB p65 pathway in rat glomerular mesangial cells under high glucose conditions, leading to upregulation of miR-155-5p, and downstream increases in CTGF and fibronectin (fibrosis markers). TNC knockdown reduced TLR4, p-NF-κB p65, miR-155-5p, CTGF, and fibronectin levels. Metformin treatment reduced TNC, p-NF-κB p65, CTGF, and FN protein levels.","method":"TNC knockdown in rat mesangial cells, high glucose stimulation, Western blot, qPCR, ELISA for TNC, immunohistochemistry in diabetic rat glomeruli","journal":"World Journal of Diabetes","confidence":"Medium","confidence_rationale":"Tier 3 — siRNA knockdown with multiple downstream readouts in single study, extending TLR4 mechanism to kidney fibrosis","pmids":["33520106"],"is_preprint":false},{"year":2021,"finding":"miR-218 targets the 3'UTR of TNC mRNA (confirmed by dual-luciferase reporter assay), suppressing TNC protein expression. Loss of TNC reduces AKT phosphorylation and JNK phosphorylation (AP-1 transcriptional activity), which decreases TGFβ1 expression. TGFβ1 in turn activates the TNC/AKT/AP-1 axis, forming a positive feedback loop. miR-218 overexpression inhibited glioma cell proliferation, migration, invasion, and tumor growth in nude mice, and induced apoptosis.","method":"Dual-luciferase reporter (3'UTR binding), Western blot (AKT/JNK phosphorylation), miR-218 overexpression/inhibition, in vivo nude mouse xenograft, colony formation, invasion assays","journal":"International Journal of Molecular Medicine","confidence":"Medium","confidence_rationale":"Tier 2–3 — 3'UTR luciferase confirmation of direct targeting, combined with in vitro and in vivo functional studies, single lab","pmids":["34558654"],"is_preprint":false},{"year":2024,"finding":"Transcription factor Twist1 promotes kidney fibrosis by driving TNC expression through an indirect mechanism: Twist1 directly binds the Prrx1 promoter to upregulate Prrx1, and Prrx1 in turn directly binds the TNC promoter to upregulate TNC expression. Fibroblast-specific Twist1 knockout mice showed reduced Prrx1 and TNC protein, decreased interstitial ECM deposition, and less kidney inflammation in UUO and ischemia-reperfusion fibrosis models. TGF-β1 induces Twist1 in fibroblasts.","method":"Chromatin immunoprecipitation (Twist1 binding to Prrx1 promoter; Prrx1 binding to TNC promoter), fibroblast-specific Twist1 KO mice (UUO and IR injury models), TGF-β1 stimulation, gain/loss-of-function experiments","journal":"Kidney International","confidence":"High","confidence_rationale":"Tier 1–2 — ChIP establishing direct promoter binding at two steps in the pathway, validated in vivo with fibroblast-specific KO in two injury models","pmids":["39181396"],"is_preprint":false},{"year":2025,"finding":"CREB5, activated by endoplasmic reticulum stress (ERS) via a super-enhancer, directly binds the TNC promoter to upregulate TNC transcription, thereby promoting epithelial-mesenchymal transition (EMT) in hepatocellular carcinoma cells. ChIP-seq identified the ERS-related super-enhancer, and CREB5 overexpression increased TNC expression and EMT markers; CREB5 knockdown reduced them. This CREB5/TNC axis is activated downstream of ERS.","method":"ChIP-seq (super-enhancer identification), ChIP (CREB5 binding to TNC promoter), RT-qPCR, Western blot, CRISPR-Cas9 KO and overexpression, TCGA dataset analysis, tissue arrays","journal":"Cell Death & Disease","confidence":"Medium","confidence_rationale":"Tier 1–2 — ChIP-seq + ChIP establishing direct promoter binding, with functional overexpression/knockdown, but single study","pmids":["39915455"],"is_preprint":false},{"year":2019,"finding":"miR-9-5p directly targets and suppresses Tnc (tenascin-C) expression in chondrocytes; the miR-9-5p binding site on Tnc was confirmed by luciferase reporter assay. In an OA mouse model following tibial plateau fracture, miR-9-5p was downregulated while Tnc was upregulated. Overexpression of miR-9-5p or Tnc knockdown inhibited chondrocyte apoptosis and promoted proliferation, migration, and cartilage remodeling.","method":"Luciferase reporter assay (miR-9-5p/Tnc 3'UTR), gain/loss-of-function in OA mouse model and in vitro, Western blot, Mankin scoring of cartilage","journal":"Journal of Cellular Physiology","confidence":"Medium","confidence_rationale":"Tier 3 — luciferase reporter plus in vivo and in vitro functional studies, single lab","pmids":["31169312"],"is_preprint":false}],"current_model":"Tenascin-C (TNC) is a multidomain extracellular matrix glycoprotein that functions as an endogenous TLR4 activator driving inflammation and fibrosis, signals through multiple integrin receptors (αvβ3, α2β1, α8β1, α9β1) and annexin II (via its alternatively spliced segment) to modulate cell adhesion and RhoA/Rac-dependent invasion, directly activates EGFR via its EGF-like repeats, is incorporated into ECM meshworks by periostin, has its expression transcriptionally regulated by HIF1α (downstream of IDH1 and tissue stiffness), Twist1/Prrx1, CREB5, and miR-218/miR-9-5p, and its FNIII domain structure has been resolved crystallographically at 1.8 Å; collectively these mechanisms link TNC to arthritis, fibrosis, cancer invasion, and tissue remodeling."},"narrative":{"teleology":[{"year":1991,"claim":"Establishing the complete modular architecture of human TNC—including 14.5 EGF-like repeats, 15 FNIII repeats, and alternatively spliced isoforms—provided the domain framework necessary for all subsequent receptor-mapping and signaling studies.","evidence":"cDNA sequencing with PCR-based splice variant identification and monoclonal antibody epitope mapping","pmids":["1707164"],"confidence":"High","gaps":["No functional assays for individual domains at this stage","Stoichiometry of isoform expression across tissues not determined"]},{"year":1992,"claim":"Solving the FNIII domain crystal structure at 1.8 Å revealed the immunoglobulin-superfamily fold and exposed RGD loop, explaining how TNC presents cell-adhesion motifs and providing the first atomic-level understanding of an ECM repeat module.","evidence":"X-ray crystallography with MAD phasing of selenomethionyl protein","pmids":["1279805"],"confidence":"High","gaps":["Only one of 15 FNIII repeats was structurally resolved","No structures of EGF-like repeats or the fibrinogen-like globe"]},{"year":1993,"claim":"Identification of multiple integrin receptors (αvβ3 via RGD, α2β1 via a non-RGD site) revealed that TNC engages the cell surface through mechanistically distinct, partially redundant adhesion pathways rather than a single integrin.","evidence":"Recombinant FNIII fragment adhesion assays, RGD mutagenesis, anti-integrin blocking antibodies, and affinity purification of integrins on TNC columns in endothelial cells","pmids":["7694284","7693733"],"confidence":"High","gaps":["Structural basis of α2β1 binding to TNC not defined","Relative contribution of each integrin in vivo not determined"]},{"year":1994,"claim":"Discovery that α9β1 binds TNC's third FNIII repeat at a non-RGD site, and that annexin II serves as a high-affinity receptor for the alternatively spliced FNIII segment, expanded the receptor repertoire beyond classical integrins and demonstrated isoform-specific signaling potential.","evidence":"α9 cDNA transfection with RGD mutagenesis (α9β1); radioligand Scatchard analysis, affinity chromatography, and anti-annexin II blocking (annexin II)","pmids":["7523411","7518469"],"confidence":"High","gaps":["Downstream signaling from α9β1–TNC interaction not characterized","In vivo relevance of annexin II–TNC interaction not tested"]},{"year":1995,"claim":"Identification of α8β1 as an additional TNC integrin receptor reinforced the emerging picture of TNC as a multi-integrin ligand and showed that among RGD-containing ECM proteins, TNC has an unusually broad integrin-binding repertoire.","evidence":"α8 cDNA transfection in 293 cells, adhesion assays on recombinant TNC fragments, GRGDSP peptide elution from affinity columns","pmids":["7559467"],"confidence":"High","gaps":["Functional consequences of α8β1–TNC binding in tissue contexts undefined"]},{"year":1997,"claim":"Demonstrating that TNC–αvβ3 interaction redistributes actin and clusters EGFR, increasing its tyrosine phosphorylation, established the first mechanistic link between TNC-mediated adhesion and growth factor receptor transactivation.","evidence":"Exogenous TNC on SMCs, β3-integrin cross-linking, EGFR phosphorylation assays, actin/EGFR colocalization by immunofluorescence","pmids":["9314546"],"confidence":"High","gaps":["Whether direct EGFR binding by TNC contributes was not tested","In vivo relevance in vascular remodeling not shown"]},{"year":2001,"claim":"Two discoveries redefined TNC's signaling capacity: its EGF-like repeats were shown to directly bind and activate EGFR (functioning as a matrix-tethered growth factor), and its binding to fibronectin's FNIII13 was shown to disrupt syndecan-4 coreceptor function, explaining TNC's anti-adhesive and pro-proliferative effects on the fibronectin matrix.","evidence":"EGFR autophosphorylation, ERK activation, and bead-tethered adhesion assays with EGF competition (EGFR); syndecan-4 rescue and FNIII13 competition on mixed FN/TNC substrata (syndecan-4)","pmids":["11470832","11731446"],"confidence":"High","gaps":["Affinity of EGF-like repeats for EGFR much lower than EGF—physiological relevance of soluble presentation uncertain","Whether syndecan-4 displacement occurs in vivo not demonstrated"]},{"year":2004,"claim":"Placing TNC upstream of RhoA inactivation (via EGFR) and permissive for HGF/Rac-driven invasion established an epistatic pathway by which stromal TNC directs tumor cell morphological switching and invasive behavior.","evidence":"Collagen I/Matrigel invasion assays with dominant-negative/constitutively active Rho and Rac mutants, pharmacological inhibitors, myofibroblast co-culture","pmids":["15059978"],"confidence":"High","gaps":["In vivo invasion assays not performed","Whether TNC acts solely through EGFR-EGF-like repeats or also through integrins in this context is unresolved"]},{"year":2009,"claim":"Identification of TNC as an endogenous TLR4 ligand—via its fibrinogen-like domain—fundamentally reframed TNC from a structural/adhesion molecule to a danger-associated molecular pattern (DAMP) that sustains chronic inflammation, as demonstrated by protection of Tnc-null mice from erosive arthritis and the role of periostin in incorporating TNC into ECM meshworks.","evidence":"Tnc-KO mice in arthritis model, TLR4-blocking antibodies and TLR4-deficient cells, human synovial cell stimulation (TLR4); periostin-null and TNC-null mouse phenocopying, domain-mapping binding assays (periostin)","pmids":["19561617","19887451"],"confidence":"High","gaps":["Crystal structure of TNC fibrinogen-like domain bound to TLR4 not available","Whether periostin-mediated TNC incorporation amplifies TLR4 signaling not directly tested"]},{"year":2016,"claim":"Extension of the TLR4 mechanism to organ fibrosis (skin and lung) confirmed TNC as a general DAMP maintaining fibrosis amplification loops, while discovery of HIF1α-dependent transcriptional control downstream of IDH1 and tissue stiffness revealed a mechanosensitive feed-forward circuit in glioblastoma.","evidence":"TNC-KO mice in bleomycin skin/lung fibrosis models with fibroblast TLR4 signaling assays (fibrosis); IDH1 gain/loss-of-function xenografts with AFM stiffness measurements and HIF1α/miR-203 manipulation (glioblastoma)","pmids":["27256716","27820599"],"confidence":"High","gaps":["Whether TLR4-TNC signaling is the sole driver or synergizes with integrin/EGFR pathways in fibrosis is unclear","Quantitative threshold of ECM stiffness required to activate HIF1α-TNC loop not defined"]},{"year":2019,"claim":"miR-9-5p was validated as a direct post-transcriptional suppressor of TNC in chondrocytes, linking miRNA dysregulation to TNC-driven cartilage pathology in osteoarthritis.","evidence":"Luciferase reporter assay confirming miR-9-5p binding to Tnc 3'UTR, gain/loss-of-function in OA mouse model","pmids":["31169312"],"confidence":"Medium","gaps":["Single study from one lab","Whether miR-9-5p regulation of TNC operates in tissues beyond cartilage is unknown"]},{"year":2021,"claim":"miR-218 was confirmed as a second direct miRNA repressor of TNC, and TNC was placed within a TGFβ1/AKT/AP-1 positive feedback loop in glioma, further elucidating post-transcriptional control and downstream signaling.","evidence":"Dual-luciferase 3'UTR reporter, miR-218 overexpression/inhibition, nude mouse xenograft","pmids":["34558654"],"confidence":"Medium","gaps":["Relative importance of miR-218 vs miR-9-5p in controlling TNC levels in different tissues not compared","Feedback loop not validated in non-glioma contexts"]},{"year":2024,"claim":"ChIP-based demonstration that Twist1 drives TNC expression indirectly through Prrx1 in fibroblasts provided the first two-step transcription factor cascade for TNC in kidney fibrosis, validated with fibroblast-specific Twist1 knockout mice.","evidence":"ChIP for Twist1 on Prrx1 promoter and Prrx1 on TNC promoter, fibroblast-specific Twist1-KO mice in UUO and IR injury","pmids":["39181396"],"confidence":"High","gaps":["Whether Twist1→Prrx1→TNC cascade operates in fibrosis of other organs not tested","Direct versus Prrx1-mediated contribution of Twist1 not quantified"]},{"year":2025,"claim":"Discovery that ER stress activates CREB5 via a super-enhancer to directly drive TNC transcription and EMT in hepatocellular carcinoma added a stress-responsive transcriptional input to the growing regulatory network.","evidence":"ChIP-seq for super-enhancer, ChIP for CREB5 on TNC promoter, CRISPR-Cas9 KO and overexpression","pmids":["39915455"],"confidence":"Medium","gaps":["Single study; independent replication needed","Whether CREB5-driven TNC activates TLR4 or EGFR in this context not examined"]},{"year":null,"claim":"Key unresolved questions include: the structural basis of TNC fibrinogen-like domain recognition by TLR4; how multiple receptor systems (integrins, EGFR, TLR4, annexin II) are spatially and temporally coordinated on a single hexabrachion; and the in vivo hierarchy among transcriptional (HIF1α, Twist1/Prrx1, CREB5) and post-transcriptional (miR-218, miR-9-5p) regulatory inputs across different tissue and disease contexts.","evidence":"","pmids":[],"confidence":"Low","gaps":["No co-crystal structure of TNC-TLR4 or TNC-EGFR","Relative signaling output from simultaneous engagement of multiple receptor types on a hexabrachion not quantified","Integrated regulatory model across tissues lacking"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0098631","term_label":"cell adhesion mediator activity","supporting_discovery_ids":[2,3,5,6]},{"term_id":"GO:0048018","term_label":"receptor ligand activity","supporting_discovery_ids":[8,11,16]},{"term_id":"GO:0005198","term_label":"structural molecule activity","supporting_discovery_ids":[0,1,12]}],"localization":[{"term_id":"GO:0031012","term_label":"extracellular matrix","supporting_discovery_ids":[0,1,9,12]},{"term_id":"GO:0005576","term_label":"extracellular region","supporting_discovery_ids":[4,11,16]}],"pathway":[{"term_id":"R-HSA-1474244","term_label":"Extracellular matrix organization","supporting_discovery_ids":[0,1,9,12]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[11,16,21]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[7,8,10,17]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[10,17,22]}],"complexes":[],"partners":["EGFR","ITGAV","ITGB3","ITGA2","ITGB1","ITGA9","ANXA2","POSTN"],"other_free_text":[]},"mechanistic_narrative":"Tenascin-C (TNC) is a hexabrachion-forming extracellular matrix glycoprotein that orchestrates cell adhesion, inflammation, fibrosis, and tissue remodeling by engaging multiple receptor systems and signaling pathways. Its modular architecture—comprising EGF-like repeats, alternatively spliced fibronectin type III (FNIII) domains, and a fibrinogen-like globe—enables distinct receptor interactions: EGF-like repeats directly activate EGFR to stimulate mitogenesis and RhoA inactivation favoring invasion [PMID:11470832, PMID:15059978]; FNIII domains engage integrins α2β1, αvβ3, α8β1, and α9β1 through RGD-dependent and -independent mechanisms [PMID:7694284, PMID:7693733, PMID:7523411, PMID:7559467]; the alternatively spliced FNIII segment binds annexin II with nanomolar affinity [PMID:7518469]; and the fibrinogen-like domain activates TLR4 to drive NF-κB–dependent cytokine production, sustaining chronic inflammation in arthritis and organ fibrosis [PMID:19561617, PMID:27256716]. TNC expression is transcriptionally controlled by HIF1α (downstream of IDH1 status and tissue stiffness), Twist1→Prrx1, and CREB5, and post-transcriptionally repressed by miR-218 and miR-9-5p [PMID:27820599, PMID:39181396, PMID:39915455, PMID:34558654, PMID:31169312]. Missense mutations in the fibrinogen-like domain co-segregate with autosomal dominant nonsyndromic hearing loss (DFNA56) [PMID:23936043]."},"prefetch_data":{"uniprot":{"accession":"P24821","full_name":"Tenascin","aliases":["Cytotactin","GMEM","GP 150-225","Glioma-associated-extracellular matrix antigen","Hexabrachion","JI","Myotendinous antigen","Neuronectin","Tenascin-C","TN-C"],"length_aa":2201,"mass_kda":240.9,"function":"Extracellular matrix protein implicated in guidance of migrating neurons as well as axons during development, synaptic plasticity as well as neuronal regeneration. Promotes neurite outgrowth from cortical neurons grown on a monolayer of astrocytes. Ligand for integrins alpha-8/beta-1, alpha-9/beta-1, alpha-V/beta-3 and alpha-V/beta-6. 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CMH13","url":"https://www.omim.org/entry/613243"},{"mim_id":"613207","title":"ASTHMA-RELATED TRAITS, SUSCEPTIBILITY TO, 8; ASRT8","url":"https://www.omim.org/entry/613207"},{"mim_id":"611768","title":"MICRO RNA 335; MIR335","url":"https://www.omim.org/entry/611768"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in all","driving_tissues":[{"tissue":"blood vessel","ntpm":345.5},{"tissue":"smooth 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chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/7559467","citation_count":189,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"28675934","id":"PMC_28675934","title":"Characterization of the Extracellular Matrix of Normal and Diseased Tissues Using Proteomics.","date":"2017","source":"Journal of proteome research","url":"https://pubmed.ncbi.nlm.nih.gov/28675934","citation_count":185,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"7693733","id":"PMC_7693733","title":"Endothelial cell attachment and spreading on human tenascin is mediated by alpha 2 beta 1 and alpha v beta 3 integrins.","date":"1993","source":"Journal of cell science","url":"https://pubmed.ncbi.nlm.nih.gov/7693733","citation_count":185,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"7523411","id":"PMC_7523411","title":"The integrin alpha 9 beta 1 mediates cell attachment to a non-RGD site in the third fibronectin type III repeat of tenascin.","date":"1994","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/7523411","citation_count":182,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"21653829","id":"PMC_21653829","title":"Protein interactome reveals converging molecular pathways among autism disorders.","date":"2011","source":"Science translational medicine","url":"https://pubmed.ncbi.nlm.nih.gov/21653829","citation_count":180,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"22261194","id":"PMC_22261194","title":"Proteomics analysis of cardiac extracellular matrix remodeling in a porcine model of ischemia/reperfusion injury.","date":"2012","source":"Circulation","url":"https://pubmed.ncbi.nlm.nih.gov/22261194","citation_count":179,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"8824254","id":"PMC_8824254","title":"Binding of the NG2 proteoglycan to type VI collagen and other extracellular matrix molecules.","date":"1996","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/8824254","citation_count":176,"is_preprint":false,"source_track":"gene2pubmed"}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":50935,"output_tokens":3708,"usd":0.104212},"stage2":{"model":"claude-opus-4-6","input_tokens":6416,"output_tokens":2740,"usd":0.15087},"total_usd":0.655107,"stage1_batch_id":"msgbatch_01RPbVc9PDH9zQmYK5Hs6srm","stage2_batch_id":"msgbatch_01JVbVLzeKN2yVrsGp3zC8aM","note":"batch pricing = 50% of standard","round2_stage1":{"model":"claude-sonnet-4-6","input_tokens":65233,"output_tokens":8845,"usd":0.164187},"round2_rules_fired":"R3","round2_stage2":{"model":"claude-opus-4-6","input_tokens":11585,"output_tokens":3972,"usd":0.235838}},"stage1_raw":"Looking at the abstracts, I need to identify which papers are about the human gene TNC (tenascin-C), an extracellular matrix glycoprotein. I need to distinguish this from other uses of \"TNC\" (e.g., Tn antigen/Tn syndrome, Tn-seq bacterial methods, troponin C abbreviation \"TN-C\", trigeminal nucleus caudalis \"TNC\", trophoblastic neoplasia \"TN\", etc.).\n\nRelevant papers about tenascin-C (TNC) as an ECM protein:\n- PMID:23936043 (hearing loss, TNC/tenascin-C in cochlea)\n- PMID:32011035 (TNC-USP6 fusion in aneurysmal bone cyst)\n- PMID:21298289 (TNC polymorphisms and atherosclerosis)\n- PMID:22045641 (TNC promoter activity, cell cycle)\n- PMID:18751374 (EMMPRIN modulates TN-C deposition in oral squamous carcinoma)\n- PMID:24598996 (mTOR/TNC FNIII A1 in osteosarcoma)\n- PMID:39181396 (Twist1/Prrx1/TNC in kidney fibrosis)\n- PMID:39915455 (CREB5/TNC in HCC EMT)\n- PMID:34558654 (miR-218/TNC/AKT/AP-1/TGFβ1 in glioma)\n- PMID:31169312 (miR-9-5p/Tnc in OA chondrocytes)\n- PMID:33520106 (TNC/TLR4/NF-κB/miR-155-5p in DKD)\n- PMID:40598496 (TNC/EndMT/CAFs in GBM)\n- PMID:9253598 (NRAMP1 and TNC in chicken Salmonella resistance - TNC here refers to the tenascin-C locus linked to Lps)\n- PMID:7530615 (swine tenascin gene HXB chromosomal location)\n- PMID:23192621 (TNC variants and Achilles tendinopathy)\n- PMID:23906005 (TNC/COL5A1 and tendinopathy in horses)\n- PMID:30528246 (TNC variants and ACL rupture)\n- PMID:88 (mTOR/TNC in osteosarcoma - already noted)\n\nNow let me extract mechanistic discoveries:\n\n```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2008,\n      \"finding\": \"EMMPRIN (extracellular matrix metalloproteinase inducer) regulates tenascin-C (TN-C) matrix deposition in oral squamous cell carcinoma: overexpression of EMMPRIN in poorly invasive SCC cells increased TN-C deposition in SCC/peritumoral fibroblast co-cultures several-fold, while siRNA knockdown of EMMPRIN reduced TN-C deposition to minimal levels. MMP inhibition with GM6001 suppressed TN-C deposition, indicating MMP activity is required downstream of EMMPRIN for TN-C matrix assembly.\",\n      \"method\": \"Retroviral overexpression and siRNA knockdown of EMMPRIN in SCC cells; co-culture with peritumoral fibroblasts; broad-spectrum MMP inhibitor (GM6001) treatment; TN-C deposition measured by immunoassay\",\n      \"journal\": \"Anticancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — clean gain/loss-of-function with defined cellular phenotype, replicated across OE and KD conditions; single lab\",\n      \"pmids\": [\"18751374\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"TNC promoter activity is dynamically regulated through the cell cycle: live-cell imaging of NIH 3T3 cells stably expressing a TNC promoter-driven destabilized GFP reporter showed that TNC promoter activity increases on average during the last 40% of the cell cycle, and that the magnitude of this increase is proportional to promoter activity earlier in the cycle.\",\n      \"method\": \"Stable transfection of TNC promoter-dGFP reporter in NIH 3T3 cells; automated live-cell fluorescence and phase-contrast microscopy over 62 h; single-cell quantification across hundreds of cells\",\n      \"journal\": \"Cytometry. Part A\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct live-cell localization/activity measurement with functional link to cell cycle; single lab, single method\",\n      \"pmids\": [\"22045641\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Missense mutations in TNC (encoding tenascin-C) cause autosomal dominant nonsyndromic progressive hearing loss (DFNA56). Two co-segregating mutations (p.V1773M and p.T1796S) were identified in conserved regions of TNC in two unrelated Chinese families with hereditary hearing loss, consistent with tenascin-C's established localization to the basilar membrane and osseous spiral lamina of the cochlea and its role in cochlear development.\",\n      \"method\": \"Exome sequencing in affected family members; linkage analysis mapping to chromosome 9q31.3-q34.3; Sanger sequencing and mass spectrometry (Sequenom) co-segregation analysis in 53 subjects; in silico SIFT prediction of deleteriousness; screening of 587 NSHL subjects\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — genetic co-segregation across two independent families with functional context from known cochlear localization; no in vitro mechanistic rescue experiment\",\n      \"pmids\": [\"23936043\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Mechanical stress upregulates expression of the alternatively spliced tenascin-C FNIII A1 domain in osteosarcoma (MG-63) cells via the mTOR signaling pathway, with downstream molecules 4E-BP1 and S6K1 required for both basal and mechanically induced A1 expression. TNC FNIII A1 overexpression promotes MG-63 cell migration.\",\n      \"method\": \"Mechanical stimulation of MG-63 osteosarcoma cells; mTOR inhibition; qPCR and western blot for A1 mRNA and protein; cell migration assay; immunohistochemistry of osteosarcoma tissues\",\n      \"journal\": \"Molecules and cells\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — multiple methods (migration assay, signaling inhibition, expression analysis) establishing pathway; single lab\",\n      \"pmids\": [\"24598996\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"TNC (tenascin-C) forms novel gene fusions with USP6 in primary aneurysmal bone cysts, identified as TNC-USP6 fusions in two ABC cases by next-generation sequencing, confirmed by RT-PCR and Sanger sequencing.\",\n      \"method\": \"Next-generation sequencing (NGS) of 11 consecutive aneurysmal bone cysts; RT-PCR and Sanger sequencing confirmation of TNC-USP6 fusion\",\n      \"journal\": \"Genes, chromosomes & cancer\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — molecular identification of fusion by NGS with orthogonal confirmation; two independent cases; single lab\",\n      \"pmids\": [\"32011035\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"TNC activates an inflammatory and fibrotic signaling loop in rat mesangial cells under high-glucose conditions via the TLR4/NF-κB p65/miR-155-5p pathway: TNC knockdown reduced TLR4, phospho-NF-κB p65, miR-155-5p, CTGF, and fibronectin levels, identifying TNC as an upstream activator of this inflammatory cascade in diabetic kidney disease.\",\n      \"method\": \"TNC knockdown in rat glomerular mesangial cells under high-glucose stimulation; western blot and qPCR for TLR4, p-NF-κB p65, miR-155-5p, CTGF, fibronectin; ELISA for serum/tissue TNC; immunohistochemistry in DKD rat kidneys\",\n      \"journal\": \"World journal of diabetes\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — loss-of-function with defined pathway readout across multiple markers; single lab\",\n      \"pmids\": [\"33520106\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"miR-218 suppresses glioma cell malignancy by directly binding the 3′-UTR of TNC mRNA and reducing TNC expression, which reduces AKT phosphorylation and inhibits AP-1 transcriptional activity (via reduced JNK phosphorylation), downregulating TGFβ1. TGFβ1 in turn activates the TNC/AKT/AP-1 axis, forming a positive feedback loop that miR-218 disrupts.\",\n      \"method\": \"Dual-luciferase reporter assay confirming miR-218 binding to TNC 3′-UTR; western blot for TNC, p-AKT, p-JNK, AP-1, TGFβ1; in vitro proliferation, migration, invasion, colony formation assays; in vivo nude mouse tumorigenicity assay\",\n      \"journal\": \"International journal of molecular medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1–2 — luciferase reporter validates direct miR-218/TNC interaction; western blot pathway analysis with multiple readouts; in vivo confirmation; single lab\",\n      \"pmids\": [\"34558654\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Twist1 transcription factor drives kidney fibroblast activation and kidney fibrosis by directly binding the Prrx1 promoter to upregulate Prrx1, which in turn directly binds the TNC promoter to drive tenascin-C expression. Fibroblast-specific Twist1 knockout in mice reduced Prrx1 and TNC protein abundance and decreased interstitial ECM deposition and inflammation in two kidney injury models (UUO and IRI).\",\n      \"method\": \"ChIP (Twist1 binding to Prrx1 promoter; Prrx1 binding to TNC promoter); gain- and loss-of-function experiments in rat kidney interstitial fibroblasts with TGF-β1; fibroblast-specific Twist1 conditional knockout mice; unilateral ureteral obstruction and ischemia-reperfusion injury kidney fibrosis models; western blot, immunohistochemistry\",\n      \"journal\": \"Kidney international\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — ChIP establishes direct transcriptional hierarchy Twist1→Prrx1→TNC; genetic KO with defined in vivo phenotype; multiple orthogonal methods\",\n      \"pmids\": [\"39181396\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"CREB5 transcription factor directly binds the TNC promoter and upregulates tenascin-C expression to promote epithelial-mesenchymal transition (EMT) in hepatocellular carcinoma cells downstream of endoplasmic reticulum stress-activated super-enhancers.\",\n      \"method\": \"ChIP-seq and RNA-seq identifying ERS-related super-enhancers; CRISPR-Cas9 identification of CREB5 as SE target; ChIP of CREB5 binding to TNC promoter; CREB5 overexpression/knockdown with EMT marker readouts; in vitro proliferation, migration, invasion, apoptosis assays\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1–2 — ChIP validates direct CREB5 binding to TNC promoter; functional gain/loss-of-function; single lab\",\n      \"pmids\": [\"39915455\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"miR-9-5p directly targets Tnc (tenascin-C) mRNA in mouse chondrocytes; overexpression of miR-9-5p reduced Tnc expression and inhibited chondrocyte apoptosis while promoting proliferation, migration, invasion, and cartilage remodeling in a mouse osteoarthritis model following tibial plateau fracture.\",\n      \"method\": \"Target validation (Tnc confirmed as miR-9-5p target gene); gain- and loss-of-function experiments in chondrocytes; in vivo OA mouse model with Mankin score; collagen type II/X and protogenin expression; cell proliferation, migration, invasion, apoptosis assays\",\n      \"journal\": \"Journal of cellular physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — target validation with multiple cellular and in vivo readouts; single lab\",\n      \"pmids\": [\"31169312\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"Tenascin-C (TNC) is an extracellular matrix glycoprotein whose expression is transcriptionally regulated by a Twist1→Prrx1→TNC promoter-binding hierarchy in fibroblasts and by CREB5 binding to its promoter under ER stress, and whose deposition is controlled upstream by EMMPRIN-dependent MMP activity; TNC in turn activates TLR4/NF-κB/miR-155-5p inflammatory signaling, promotes AKT/AP-1/TGFβ1-mediated malignancy (regulated by miR-218), is upregulated by mechanical stress via mTOR/4E-BP1/S6K1 signaling to promote cell migration, is targeted by miR-9-5p to modulate chondrocyte survival, and forms oncogenic TNC-USP6 gene fusions in aneurysmal bone cyst, while loss-of-function mutations in its conserved domains cause autosomal dominant hearing loss.\"\n}\n```\n\n--- ROUND 2 (R2/R3, augmented corpus) ---\n\nI'll systematically classify each paper, then extract discoveries from KEEP papers only.\n\n**Classification of papers about TNC (Tenascin-C):**\n\nMost papers in the retrieved corpus are about Tn antigen (tumor-associated carbohydrate antigen GalNAc-O-Ser/Thr), Tn-seq (transposon sequencing), TNC as abbreviation for trigeminal nucleus caudalis, TN-C as troponin C, or other unrelated uses. These are EXCLUDE (alias collisions).\n\nKEEP papers (canonical TNC = Tenascin-C protein):\n- PMID:23936043 - TNC gene and hearing loss\n- PMID:19561617 - Tenascin-C activates TLR4 in arthritis\n- PMID:10842355 - Tenascin family structure/function review\n- PMID:15059978 - TNC and colon cancer invasion via RhoA/Rac\n- PMID:9314546 - TNC, αvβ3 integrin, EGFR signaling\n- PMID:27820599 - IDH1-HIF1α-TNC feedback in glioblastoma\n- PMID:27256716 - TNC drives fibrosis via TLR4\n- PMID:11470832 - EGF-like repeats of TNC as EGFR ligands\n- PMID:19887451 - Periostin incorporates TNC into ECM\n- PMID:11731446 - TNC interference with syndecan-4/fibronectin\n- PMID:7694284 - Multiple integrins mediate attachment to TNC (cytotactin)\n- PMID:7518469 - Annexin II as receptor for alternatively spliced TNC\n- PMID:7559467 - α8β1 integrin as TNC receptor\n- PMID:7523411 - α9β1 integrin binds non-RGD site in TNC FNIII repeat 3\n- PMID:7693733 - α2β1 and αvβ3 mediate endothelial attachment to TNC\n- PMID:1279805 - Crystal structure of TNC fibronectin type III domain\n- PMID:1707164 - Human TNC primary structure and splicing\n- PMID:22045641 - TNC promoter cell cycle activity\n- PMID:12845616 - Tenascins in pathological stress (review)\n- PMID:20551380 - Proteomics of aorta ECM (identifies TNC)\n- PMID:23192621 - TNC SNPs and Achilles tendinopathy\n- PMID:21298289 - TNC polymorphisms and CAD/atherosclerosis\n- PMID:32011035 - TNC-USP6 fusion in aneurysmal bone cyst\n- PMID:24598996 - mTOR/4E-BP1/S6K1 regulate TNC FNIIIA1 under mechanical stress\n- PMID:39181396 - Twist1/Prrx1/TNC pathway in kidney fibrosis\n- PMID:34558654 - miR-218/TNC/AKT/AP-1/TGFβ1 loop in glioma\n- PMID:31169312 - miR-9-5p targets Tnc in osteoarthritis\n- PMID:33520106 - TNC/TLR4/NF-κB/miR-155-5p in diabetic kidney\n- PMID:39915455 - CREB5/TNC in HCC EMT\n- PMID:18751374 - EMMPRIN regulates TNC deposition in SCC\n- PMID:40598496 - TNC/FLNC in glioblastoma resistance\n- PMID:8824254 - NG2 proteoglycan binds tenascin (among others)\n- PMID:21653829 - Protein interactome in ASD (TNC connectivity)\n- PMID:25037231 - ECM signatures in colon cancer (TNC identified)\n- PMID:22261194 - Cardiac ECM remodeling (TNC identified)\n- PMID:17584833 - TNC expression after muscle exercise\n- PMID:28445763 - TnC-A8V (cardiac troponin C) — EXCLUDE (troponin C, not tenascin-C)\n- PMID:123476 - TN-C cardiac troponin C — EXCLUDE\n- PMID:127652 - TN-I cardiac troponin — EXCLUDE\n- PMID:6229473 - TNC-T thymic nurse cells — EXCLUDE\n- PMID:30528246 - TNC variants and ACL — association study, no mechanism\n- PMID:23906005 - TNC polymorphisms and tendinopathy — association, no mechanism\n- PMID:7530615 - Swine tenascin gene chromosomal location — KEEP (direct experiment)\n- PMID:9253598 - Chicken NRAMP1 and TNC in Salmonella resistance — KEEP (genetic linkage)\n\nNow extracting mechanistic discoveries from KEEP papers:\n\n```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1992,\n      \"finding\": \"The crystal structure of a fibronectin type III (FNIII) domain from tenascin-C was solved by multiwavelength anomalous diffraction (MAD) phasing of the selenomethionyl protein to 1.8 Å resolution, revealing a folding topology identical to extracellular domains of the human growth hormone receptor and CD4, and distinct from but related to immunoglobulin constant domains. An RGD sequence mediating cell adhesion was found in a tight turn on an exposed loop.\",\n      \"method\": \"X-ray crystallography (MAD phasing, selenomethionyl protein), 1.8 Å resolution\",\n      \"journal\": \"Science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure with full atomic resolution, foundational structural paper\",\n      \"pmids\": [\"1279805\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1991,\n      \"finding\": \"The complete primary structure of human tenascin-C was established by cDNA sequencing, identifying 14.5 EGF-like repeats and 15 fibronectin type III repeats (versus 11 in chicken). At least four different isoforms containing different numbers of FNIII repeats were identified by PCR, demonstrating alternative splicing of the TNC pre-mRNA. Monoclonal antibody BC-2 was mapped to an alternatively spliced FNIII repeat, while BC-4 recognized an EGF-like epitope present in all isoforms.\",\n      \"method\": \"cDNA sequencing, PCR amplification of splice variants, expression vector library screening, epitope mapping\",\n      \"journal\": \"Nucleic Acids Research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — primary structure determination with functional domain mapping and splice variant identification\",\n      \"pmids\": [\"1707164\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1993,\n      \"finding\": \"Multiple integrins of the αv subtype mediate cell attachment to the third FNIII repeat of tenascin-C (cytotactin) containing the RGD tripeptide. However, mutation of RGD to RAD did not abolish attachment, and anti-β1 antibodies partially inhibited binding to intact TNC but not the FNIII repeat, indicating additional integrin receptors. Thus TNC engages multiple integrin receptor subtypes through partially RGD-dependent and -independent mechanisms.\",\n      \"method\": \"Recombinant fusion protein cell attachment assays, RGD peptide competition, anti-integrin blocking antibodies\",\n      \"journal\": \"Proceedings of the National Academy of Sciences\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal blocking antibody experiments with recombinant domain fragments, replicated across cell lines\",\n      \"pmids\": [\"7694284\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1993,\n      \"finding\": \"Human umbilical vein endothelial cells attach to and spread on human tenascin-C via two distinct integrin receptors: α2β1 mediates cell spreading (inhibited by anti-α2 and anti-β1 antibodies), and αvβ3 mediates partial cell attachment via the SRRGDMS site (inhibited by LM609). Both integrins were affinity-purified on tenascin-C columns. The α2β1 binding site on TNC is distinct from the RGD site.\",\n      \"method\": \"Anti-integrin blocking antibodies, affinity chromatography on TNC-coated columns, radio-receptor binding assay\",\n      \"journal\": \"Journal of Cell Science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — affinity purification combined with blocking antibodies, identifying two distinct receptor-ligand interactions\",\n      \"pmids\": [\"7693733\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1994,\n      \"finding\": \"The alternatively spliced segment of tenascin-C (TNfnA-D) binds specifically to annexin II on the cell surface of glioma and endothelial cells with ~2 nM affinity (~2–5 × 10^5 binding sites/cell). Annexin II was identified by Scatchard analysis, blot binding assays, affinity chromatography, and protein sequencing. Anti-annexin II antibodies blocked TNfnA-D binding to live cells, establishing annexin II as a cell-surface receptor for the alternatively spliced region of TNC.\",\n      \"method\": \"Radioligand binding assay, Scatchard analysis, affinity chromatography, protein sequencing, blocking antibodies\",\n      \"journal\": \"Journal of Cell Biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — multiple orthogonal methods including Scatchard binding kinetics, affinity purification, and protein sequencing\",\n      \"pmids\": [\"7518469\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1994,\n      \"finding\": \"The integrin α9β1 mediates cell attachment to the third FNIII repeat of tenascin-C at a site that is not the RGD tripeptide: RGD peptide competition and RGD→RAD/RAA mutations did not inhibit α9β1 binding. α9β1 did not mediate attachment to fibronectin, laminin, vitronectin, fibrinogen, thrombospondin, or collagens, identifying tenascin-C as a specific ligand for this integrin.\",\n      \"method\": \"Stable transfection of α9 cDNA in 293 and SW480 cells, cell adhesion assays with recombinant TNC fragments, RGD peptide competition, mutagenesis of RGD site\",\n      \"journal\": \"Journal of Biological Chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — transfection + recombinant fragment mapping + mutagenesis establishing a non-RGD binding mechanism\",\n      \"pmids\": [\"7523411\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1995,\n      \"finding\": \"The integrin α8β1 functions as a receptor for tenascin-C, fibronectin, and vitronectin. α8β1 binds to the RGD-containing third FNIII repeat of tenascin-C (eluted by GRGDSP peptide), localizes to focal contacts on fibronectin and vitronectin substrata, and mediates adhesion of α8-transfected 293 cells. Among RGD-containing ECM proteins, only tenascin was able to mediate adhesion via α8β1 in addition to fibronectin and vitronectin.\",\n      \"method\": \"Stable transfection of α8 cDNA, cell adhesion assays, affinity chromatography on fibronectin/vitronectin-Sepharose with RGD peptide elution, recombinant TNC fragment binding\",\n      \"journal\": \"Journal of Biological Chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — transfection + affinity chromatography + recombinant fragment assays\",\n      \"pmids\": [\"7559467\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1997,\n      \"finding\": \"Tenascin-C promotes survival and EGF-dependent growth of vascular smooth muscle cells (SMCs) through interaction with αvβ3 integrins. TNC-αvβ3 interaction redistributes filamentous actin to focal adhesion complexes that colocalize with EGF receptor clusters, increasing EGF-R tyrosine phosphorylation and activation. Cross-linking β3 integrins replicates TNC effects on EGF-R clustering and phosphorylation. MMP inhibition (GM6001) suppresses TNC expression; denatured collagen induces TNC in a β3-integrin-dependent manner.\",\n      \"method\": \"Exogenous TNC addition to SMCs, MMP inhibitor treatment, floating collagen gel assays, β3-integrin cross-linking, EGF-R phosphorylation assays, actin/EGF-R colocalization by immunofluorescence\",\n      \"journal\": \"Journal of Cell Biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal experiments linking TNC-αvβ3 interaction to EGF-R clustering and phosphorylation with pharmacological and antibody interventions\",\n      \"pmids\": [\"9314546\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"Select EGF-like repeats of tenascin-C directly bind to and activate the EGF receptor (EGFR), inducing EGFR autophosphorylation and ERK/MAPK activation, and stimulating mitogenesis. Micromolar concentrations of EGF-like repeats induced EGFR autophosphorylation to levels comparable to subsaturating EGF. When tethered to inert beads (simulating hexabrachion presentation), EGF-like repeats mediated EGFR-dependent adhesion. Specific EGFR binding was confirmed by immunofluorescence and EGFR cross-linking, both abolished by EGF competition.\",\n      \"method\": \"EGFR autophosphorylation assays, ERK/MAPK activation, mitogenesis assay, bead-mediated adhesion, immunofluorescence, EGF competition, EGFR cross-linking\",\n      \"journal\": \"Journal of Cell Biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — multiple biochemical assays plus competition experiments establishing TNC EGF-like repeats as direct EGFR ligands\",\n      \"pmids\": [\"11470832\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"Tenascin-C directly binds to the 13th fibronectin type III repeat (FNIII13) of fibronectin, blocking syndecan-4 binding to fibronectin's HepII site. This inhibits integrin signaling (syndecan-4 coreceptor function) and disrupts cell adhesion while promoting tumor cell proliferation. Overexpression of syndecan-4 (but not syndecan-1 or -2) rescued the adhesion defect, and addition of FNIII13 restored adhesion and normalized proliferation.\",\n      \"method\": \"Mixed fibronectin/TNC substratum adhesion assays, syndecan overexpression (rescue experiment), recombinant FNIII13 competition, cell proliferation assays\",\n      \"journal\": \"Cancer Research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — domain-specific binding mapped with recombinant fragments, validated by specific syndecan-4 rescue experiment\",\n      \"pmids\": [\"11731446\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Tenascin-C secreted by myofibroblasts promotes invasion of colon cancer cells by inactivating RhoA via the EGF-like repeats of TNC through EGFR signaling, creating a permissive signal for SF/HGF-activated Rac via c-Met. Myofibroblast-stimulated invasion is characterized by a morphological shift from round (high RhoA, low Rac) to elongated (low RhoA, high Rac) phenotype. Dominant-negative and constitutively active mutants of RhoA-ROCK and Rac, plus pharmacological modulators, confirmed this epistatic pathway: TNC acts upstream of RhoA inactivation, which is required for SF/HGF-Rac-driven invasion.\",\n      \"method\": \"In vitro invasion assay into collagen I/Matrigel, dominant-negative and constitutively active Rho/Rac mutants, pharmacological inhibitors of RhoA-ROCK and Rac, morphotype analysis, co-culture of myofibroblasts and cancer cells\",\n      \"journal\": \"FASEB Journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic epistasis via dominant-negative/constitutively active mutants plus pharmacological validation, identifying TNC's pathway position\",\n      \"pmids\": [\"15059978\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Tenascin-C is an endogenous activator of Toll-like receptor 4 (TLR4) that drives persistent synovial inflammation. Tnc-knockout mice show rapid resolution of acute joint inflammation and are protected from erosive arthritis. Intra-articular injection of TNC promotes joint inflammation in vivo. In human synovial macrophages and fibroblasts, exogenous TNC induces proinflammatory cytokine synthesis specifically via TLR4 activation. TNC's fibrinogen-like domain was identified as the TLR4-activating region.\",\n      \"method\": \"Tnc knockout mice (in vivo arthritis model), intra-articular TNC injection, explant cultures from RA synovia, human macrophage/fibroblast stimulation with TNC, TLR4-blocking antibodies and TLR4-deficient cells\",\n      \"journal\": \"Nature Medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — KO mouse in vivo model + human primary cell experiments + TLR4-blocking validation, replicated across multiple experimental systems\",\n      \"pmids\": [\"19561617\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Periostin promotes incorporation of tenascin-C into the extracellular matrix by acting as a molecular bridge: periostin possesses adjacent domains that bind to TNC and to fibronectin/type I collagen, thereby tethering TNC hexabrachions to ECM fibrils and stabilizing bifurcations to form a meshwork architecture. Both periostin-null and TNC-null mice exhibit a similar phenotype of confined tibial periostitis, and periostin deposition of TNC was demonstrated by in vitro binding assays.\",\n      \"method\": \"Periostin-null and TNC-null mouse phenotyping, in vitro protein-protein binding assays mapping periostin domains to TNC and fibronectin/collagen\",\n      \"journal\": \"Journal of Biological Chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — domain mapping by direct binding assays combined with parallel KO mouse phenotypes showing functional convergence\",\n      \"pmids\": [\"19887451\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"TNC promoter activity is dynamically regulated during the cell cycle: live cell imaging of an NIH 3T3 line stably transfected with a TNC promoter-destabilized GFP reporter showed that TNC promoter activity increases during the last 40% of the cell cycle, and that the magnitude of this increase is proportional to promoter activity earlier in the cycle.\",\n      \"method\": \"Live cell phase-contrast and fluorescence microscopy, destabilized GFP reporter under TNC promoter, automated single-cell tracking over 62 h\",\n      \"journal\": \"Cytometry Part A\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct live-cell reporter assay with single-cell resolution, but single lab, single method\",\n      \"pmids\": [\"22045641\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Missense mutations in TNC (c.5317G>A, p.V1773M and c.5368A>T, p.T1796S), located in the conserved fibrinogen-like domain, co-segregate with autosomal dominant progressive nonsyndromic hearing loss (DFNA56) in two Chinese families. TNC is expressed in the basilar membrane and osseous spiral lamina of the cochlea, implicating it in cochlear structural integrity.\",\n      \"method\": \"Genome-wide linkage analysis, whole exome sequencing, Sanger sequencing validation, mass spectrometry genotyping, segregation analysis in 53 family members\",\n      \"journal\": \"PLoS One\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — genetic segregation with predicted functional effect; no direct functional validation of mutant protein\",\n      \"pmids\": [\"23936043\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Mechanical stress upregulates expression of the alternatively spliced tenascin-C FNIIIA1 domain in osteosarcoma MG-63 cells through the mTOR/4E-BP1/S6K1 signaling pathway. Pharmacological inhibition of mTOR suppressed mechanical stress-induced A1 upregulation, and knockdown of 4E-BP1 and S6K1 reduced basal A1 expression. FNIIIA1 expression promoted MG-63 cell migration.\",\n      \"method\": \"Cyclic mechanical stress application, mTOR inhibitor treatment, siRNA knockdown of 4E-BP1 and S6K1, RT-PCR and Western blot, cell migration assay\",\n      \"journal\": \"Molecules and Cells\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — pharmacological inhibition + siRNA knockdown linking mTOR pathway to mechanically regulated TNC splicing variant expression\",\n      \"pmids\": [\"24598996\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Tenascin-C drives persistence of organ fibrosis by activating TLR4 signaling in fibroblasts. Exogenous TNC stimulates collagen gene expression and myofibroblast transformation via TLR4. Tenascin-C null mice show attenuation of both skin and lung fibrosis (bleomycin model) and accelerated fibrosis resolution. This identifies TNC as an endogenous danger signal that maintains a TLR4-dependent fibrosis amplification loop.\",\n      \"method\": \"Tenascin-C KO mice (skin and lung fibrosis models), exogenous TNC stimulation of fibroblasts, TLR4 signaling assays, collagen gene expression, myofibroblast transformation assays\",\n      \"journal\": \"Nature Communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — KO mouse models in two fibrosis contexts plus human cell mechanistic experiments, replicating and extending TLR4 mechanism from 2009 arthritis study\",\n      \"pmids\": [\"27256716\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"In glioblastoma, tissue stiffness and TNC expression form a positive feedback loop with IDH1 and HIF1α: mutant IDH1 restricts glioma aggression by reducing HIF1α-dependent TNC expression, decreasing ECM stiffness and mechanosignalling. HIF1α directly drives TNC expression; recurrent IDH1-mutant tumors bypass this suppression via reduced miR-203-mediated suppression of HIF1α and TNC. Gain- and loss-of-function xenograft manipulations confirmed that IDH1 mutation reduces TNC-ECM stiffness and tumor aggression.\",\n      \"method\": \"IDH1 gain- and loss-of-function xenograft models, atomic force microscopy (ECM stiffness), HIF1α manipulation, miR-203 functional studies, patient glioma sample analysis\",\n      \"journal\": \"Nature Cell Biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — in vivo xenograft gain/loss-of-function with AFM mechanical measurements and miRNA mechanistic studies\",\n      \"pmids\": [\"27820599\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"EMMPRIN (CD147) overexpression in oral squamous cell carcinoma (SCC) cells promotes tenascin-C deposition in co-cultures with peritumoral fibroblasts, in an MMP-dependent manner (suppressed by the broad-spectrum MMP inhibitor GM6001). EMMPRIN knockdown by siRNA reduced both cell migration and TNC deposition, while EMMPRIN overexpression increased MMP-2, -3, -9 expression and TNC deposition several-fold, establishing an EMMPRIN→MMP→TNC deposition axis.\",\n      \"method\": \"EMMPRIN retroviral overexpression, siRNA knockdown, co-culture with fibroblasts, MMP inhibitor (GM6001) treatment, TNC deposition quantification, cell migration assay\",\n      \"journal\": \"Anticancer Research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — gain and loss of function with MMP inhibitor validation in single study\",\n      \"pmids\": [\"18751374\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"NG2 proteoglycan binds to tenascin-C via its core protein (chondroitinase treatment did not reduce binding), in a concentration-dependent and saturable manner demonstrated by solid-phase binding assay. Decorin did not bind tenascin-C and could not inhibit NG2-tenascin binding, indicating NG2 uses a distinct domain for tenascin that differs from its collagen-binding region.\",\n      \"method\": \"Solid-phase binding assay with purified NG2, chondroitinase treatment, decorin competition assay\",\n      \"journal\": \"Journal of Biological Chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — direct binding assay with competition controls, single study\",\n      \"pmids\": [\"8824254\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Novel TNC-USP6 gene fusions were identified in two cases of primary aneurysmal bone cyst (ABC) by next-generation sequencing, confirmed by RT-PCR and Sanger sequencing. TNC serves as a novel fusion partner driving USP6 rearrangement in this neoplasm.\",\n      \"method\": \"Next-generation sequencing (NGS), RT-PCR, Sanger sequencing\",\n      \"journal\": \"Genes, Chromosomes & Cancer\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — NGS plus orthogonal RT-PCR/Sanger confirmation; mechanistic implication is structural (fusion driving oncogenesis), but functional consequences not directly tested\",\n      \"pmids\": [\"32011035\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"In diabetic kidney disease, TNC activates the TLR4/NF-κB p65 pathway in rat glomerular mesangial cells under high glucose conditions, leading to upregulation of miR-155-5p, and downstream increases in CTGF and fibronectin (fibrosis markers). TNC knockdown reduced TLR4, p-NF-κB p65, miR-155-5p, CTGF, and fibronectin levels. Metformin treatment reduced TNC, p-NF-κB p65, CTGF, and FN protein levels.\",\n      \"method\": \"TNC knockdown in rat mesangial cells, high glucose stimulation, Western blot, qPCR, ELISA for TNC, immunohistochemistry in diabetic rat glomeruli\",\n      \"journal\": \"World Journal of Diabetes\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — siRNA knockdown with multiple downstream readouts in single study, extending TLR4 mechanism to kidney fibrosis\",\n      \"pmids\": [\"33520106\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"miR-218 targets the 3'UTR of TNC mRNA (confirmed by dual-luciferase reporter assay), suppressing TNC protein expression. Loss of TNC reduces AKT phosphorylation and JNK phosphorylation (AP-1 transcriptional activity), which decreases TGFβ1 expression. TGFβ1 in turn activates the TNC/AKT/AP-1 axis, forming a positive feedback loop. miR-218 overexpression inhibited glioma cell proliferation, migration, invasion, and tumor growth in nude mice, and induced apoptosis.\",\n      \"method\": \"Dual-luciferase reporter (3'UTR binding), Western blot (AKT/JNK phosphorylation), miR-218 overexpression/inhibition, in vivo nude mouse xenograft, colony formation, invasion assays\",\n      \"journal\": \"International Journal of Molecular Medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — 3'UTR luciferase confirmation of direct targeting, combined with in vitro and in vivo functional studies, single lab\",\n      \"pmids\": [\"34558654\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Transcription factor Twist1 promotes kidney fibrosis by driving TNC expression through an indirect mechanism: Twist1 directly binds the Prrx1 promoter to upregulate Prrx1, and Prrx1 in turn directly binds the TNC promoter to upregulate TNC expression. Fibroblast-specific Twist1 knockout mice showed reduced Prrx1 and TNC protein, decreased interstitial ECM deposition, and less kidney inflammation in UUO and ischemia-reperfusion fibrosis models. TGF-β1 induces Twist1 in fibroblasts.\",\n      \"method\": \"Chromatin immunoprecipitation (Twist1 binding to Prrx1 promoter; Prrx1 binding to TNC promoter), fibroblast-specific Twist1 KO mice (UUO and IR injury models), TGF-β1 stimulation, gain/loss-of-function experiments\",\n      \"journal\": \"Kidney International\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — ChIP establishing direct promoter binding at two steps in the pathway, validated in vivo with fibroblast-specific KO in two injury models\",\n      \"pmids\": [\"39181396\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"CREB5, activated by endoplasmic reticulum stress (ERS) via a super-enhancer, directly binds the TNC promoter to upregulate TNC transcription, thereby promoting epithelial-mesenchymal transition (EMT) in hepatocellular carcinoma cells. ChIP-seq identified the ERS-related super-enhancer, and CREB5 overexpression increased TNC expression and EMT markers; CREB5 knockdown reduced them. This CREB5/TNC axis is activated downstream of ERS.\",\n      \"method\": \"ChIP-seq (super-enhancer identification), ChIP (CREB5 binding to TNC promoter), RT-qPCR, Western blot, CRISPR-Cas9 KO and overexpression, TCGA dataset analysis, tissue arrays\",\n      \"journal\": \"Cell Death & Disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1–2 — ChIP-seq + ChIP establishing direct promoter binding, with functional overexpression/knockdown, but single study\",\n      \"pmids\": [\"39915455\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"miR-9-5p directly targets and suppresses Tnc (tenascin-C) expression in chondrocytes; the miR-9-5p binding site on Tnc was confirmed by luciferase reporter assay. In an OA mouse model following tibial plateau fracture, miR-9-5p was downregulated while Tnc was upregulated. Overexpression of miR-9-5p or Tnc knockdown inhibited chondrocyte apoptosis and promoted proliferation, migration, and cartilage remodeling.\",\n      \"method\": \"Luciferase reporter assay (miR-9-5p/Tnc 3'UTR), gain/loss-of-function in OA mouse model and in vitro, Western blot, Mankin scoring of cartilage\",\n      \"journal\": \"Journal of Cellular Physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — luciferase reporter plus in vivo and in vitro functional studies, single lab\",\n      \"pmids\": [\"31169312\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"Tenascin-C (TNC) is a multidomain extracellular matrix glycoprotein that functions as an endogenous TLR4 activator driving inflammation and fibrosis, signals through multiple integrin receptors (αvβ3, α2β1, α8β1, α9β1) and annexin II (via its alternatively spliced segment) to modulate cell adhesion and RhoA/Rac-dependent invasion, directly activates EGFR via its EGF-like repeats, is incorporated into ECM meshworks by periostin, has its expression transcriptionally regulated by HIF1α (downstream of IDH1 and tissue stiffness), Twist1/Prrx1, CREB5, and miR-218/miR-9-5p, and its FNIII domain structure has been resolved crystallographically at 1.8 Å; collectively these mechanisms link TNC to arthritis, fibrosis, cancer invasion, and tissue remodeling.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"Tenascin-C (TNC) is an extracellular matrix glycoprotein that transduces mechanical and inflammatory signals to regulate cell migration, fibrosis, and epithelial-mesenchymal transition. Its transcription is driven by a Twist1→Prrx1→TNC promoter hierarchy in kidney fibroblasts and by CREB5 binding to its promoter under ER stress in hepatocellular carcinoma, while its matrix deposition requires upstream EMMPRIN-dependent MMP activity [PMID:39181396, PMID:39915455, PMID:18751374]. TNC activates TLR4/NF-κB/miR-155-5p inflammatory signaling in mesangial cells and sustains a TNC/AKT/AP-1/TGFβ1 positive feedback loop in glioma that is disrupted by miR-218, and mechanical stress induces its alternatively spliced FNIII A1 domain via mTOR/4E-BP1/S6K1 to promote osteosarcoma cell migration [PMID:33520106, PMID:34558654, PMID:24598996]. Loss-of-function missense mutations in conserved TNC domains cause autosomal dominant nonsyndromic progressive hearing loss (DFNA56) [PMID:23936043].\",\n  \"teleology\": [\n    {\n      \"year\": 2008,\n      \"claim\": \"The question of how TNC matrix deposition is controlled in the tumor microenvironment was answered by showing that EMMPRIN drives TNC deposition through MMP-dependent proteolytic activity, establishing an upstream regulatory axis for TNC accumulation in carcinoma stroma.\",\n      \"evidence\": \"EMMPRIN overexpression/knockdown in SCC cells co-cultured with fibroblasts, with MMP inhibitor GM6001\",\n      \"pmids\": [\"18751374\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Identity of the specific MMP(s) mediating TNC deposition not determined\", \"Whether EMMPRIN acts on TNC protein processing versus transcription not resolved\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"TNC promoter activity was shown to be cell-cycle-dependent, increasing during the last 40% of the cell cycle in proportion to earlier activity, revealing temporal regulation of TNC expression during proliferation.\",\n      \"evidence\": \"Live-cell imaging of TNC promoter–dGFP reporter in NIH 3T3 cells over 62 h with single-cell tracking\",\n      \"pmids\": [\"22045641\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Transcription factors mediating cell-cycle-dependent TNC promoter activation not identified\", \"Relevance to in vivo tissue remodeling contexts unclear\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"The question of whether TNC mutations cause human disease was addressed by identifying co-segregating missense mutations (V1773M, T1796S) in two independent families with autosomal dominant progressive hearing loss, linking TNC to DFNA56.\",\n      \"evidence\": \"Exome sequencing, linkage analysis, Sanger sequencing co-segregation in 53 subjects across two Chinese families\",\n      \"pmids\": [\"23936043\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No in vitro or in vivo functional rescue of hearing loss phenotype\", \"Structural mechanism by which mutations impair cochlear TNC function unknown\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"How mechanical stress regulates TNC was clarified: mechanical stimulation upregulates the alternatively spliced FNIII A1 domain via mTOR/4E-BP1/S6K1 signaling, and A1 overexpression promotes cell migration, establishing a mechanotransduction–TNC–migration axis.\",\n      \"evidence\": \"Mechanical stimulation of MG-63 osteosarcoma cells with mTOR pathway inhibition; qPCR, western blot, migration assay\",\n      \"pmids\": [\"24598996\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanosensor upstream of mTOR not identified\", \"Whether this axis operates in non-malignant mechano-responsive tissues not tested\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"miR-9-5p was identified as a direct post-transcriptional repressor of TNC in chondrocytes, with functional consequences for chondrocyte survival, migration, and cartilage remodeling in osteoarthritis.\",\n      \"evidence\": \"miR-9-5p target validation on Tnc; gain/loss-of-function in mouse chondrocytes and in vivo OA model\",\n      \"pmids\": [\"31169312\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Binding site mutagenesis on TNC 3′-UTR not reported\", \"Whether miR-9-5p effects are primarily TNC-dependent versus acting through additional targets unclear\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"TNC was found to participate in oncogenic gene fusions (TNC-USP6) in aneurysmal bone cysts, identifying TNC as a fusion partner that drives USP6 overexpression in this neoplasm.\",\n      \"evidence\": \"NGS of 11 ABCs with RT-PCR and Sanger sequencing confirmation in two cases\",\n      \"pmids\": [\"32011035\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Functional consequence of TNC promoter driving USP6 not experimentally demonstrated\", \"Frequency of TNC-USP6 fusion relative to other USP6 fusion partners in ABC unclear\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"TNC's role as an inflammatory signal amplifier was established: TNC activates TLR4/NF-κB p65 to induce miR-155-5p, CTGF, and fibronectin in high-glucose mesangial cells, placing TNC upstream of a defined inflammatory-fibrotic cascade in diabetic kidney disease.\",\n      \"evidence\": \"TNC knockdown in rat mesangial cells under high glucose; western blot, qPCR, ELISA; immunohistochemistry in DKD rat kidneys\",\n      \"pmids\": [\"33520106\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether TNC signals through TLR4 by direct binding not biochemically confirmed in this system\", \"Contribution of other TLR4 ligands in DKD microenvironment not controlled\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"A TNC/AKT/AP-1/TGFβ1 positive feedback loop sustaining glioma malignancy was delineated, and miR-218 was shown to disrupt it by directly targeting the TNC 3′-UTR, establishing TNC as a critical node in glioma signaling.\",\n      \"evidence\": \"Dual-luciferase reporter assay for miR-218–TNC; western blot pathway analysis; in vivo nude mouse tumorigenicity\",\n      \"pmids\": [\"34558654\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether AKT activation by TNC is receptor-mediated (e.g., integrin) or indirect not resolved\", \"Relative importance of TNC versus other miR-218 targets in glioma suppression not established\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"The transcriptional hierarchy driving TNC expression in fibroblasts was resolved: Twist1 binds the Prrx1 promoter, Prrx1 in turn binds the TNC promoter, and genetic ablation of Twist1 in fibroblasts reduces TNC and attenuates kidney fibrosis in vivo.\",\n      \"evidence\": \"ChIP for Twist1→Prrx1 and Prrx1→TNC promoter binding; fibroblast-specific Twist1 KO mice; UUO and IRI models\",\n      \"pmids\": [\"39181396\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether Prrx1 is sufficient to drive TNC in the absence of other Twist1 targets not tested\", \"Applicability to fibrosis in organs other than kidney not demonstrated\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"CREB5 was identified as a direct transcriptional activator of TNC downstream of ER-stress-activated super-enhancers, linking ER stress to TNC-driven EMT in hepatocellular carcinoma.\",\n      \"evidence\": \"ChIP-seq/RNA-seq for SE identification; ChIP of CREB5 on TNC promoter; CREB5 gain/loss-of-function with EMT readouts\",\n      \"pmids\": [\"39915455\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether TNC is the principal CREB5 effector for EMT or one of several targets not dissected\", \"In vivo validation in HCC models not reported\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The direct receptor-level mechanism by which secreted TNC activates intracellular signaling cascades (TLR4, AKT) remains incompletely defined, and whether TNC's diverse context-dependent functions reflect distinct domain-specific interactions or a common receptor engagement is unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No structural model of TNC-receptor complexes available\", \"Domain-specific contributions to signaling not systematically mapped\", \"Integration of transcriptional (Twist1/Prrx1, CREB5) and post-transcriptional (miR-218, miR-9-5p) regulation into a unified quantitative model is lacking\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [5, 6]},\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": [0, 3, 7]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0031012\", \"supporting_discovery_ids\": [0, 7]},\n      {\"term_id\": \"GO:0005576\", \"supporting_discovery_ids\": [0, 3, 5]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1474244\", \"supporting_discovery_ids\": [0, 7]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [3, 5, 6]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [5]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"PRRX1\",\n      \"TWIST1\",\n      \"CREB5\",\n      \"TLR4\",\n      \"USP6\",\n      \"BSG\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```\n\n--- ROUND 2 (R2/R3, augmented corpus) ---\n\n```json\n{\n  \"mechanistic_narrative\": \"Tenascin-C (TNC) is a hexabrachion-forming extracellular matrix glycoprotein that orchestrates cell adhesion, inflammation, fibrosis, and tissue remodeling by engaging multiple receptor systems and signaling pathways. Its modular architecture—comprising EGF-like repeats, alternatively spliced fibronectin type III (FNIII) domains, and a fibrinogen-like globe—enables distinct receptor interactions: EGF-like repeats directly activate EGFR to stimulate mitogenesis and RhoA inactivation favoring invasion [PMID:11470832, PMID:15059978]; FNIII domains engage integrins α2β1, αvβ3, α8β1, and α9β1 through RGD-dependent and -independent mechanisms [PMID:7694284, PMID:7693733, PMID:7523411, PMID:7559467]; the alternatively spliced FNIII segment binds annexin II with nanomolar affinity [PMID:7518469]; and the fibrinogen-like domain activates TLR4 to drive NF-κB–dependent cytokine production, sustaining chronic inflammation in arthritis and organ fibrosis [PMID:19561617, PMID:27256716]. TNC expression is transcriptionally controlled by HIF1α (downstream of IDH1 status and tissue stiffness), Twist1→Prrx1, and CREB5, and post-transcriptionally repressed by miR-218 and miR-9-5p [PMID:27820599, PMID:39181396, PMID:39915455, PMID:34558654, PMID:31169312]. Missense mutations in the fibrinogen-like domain co-segregate with autosomal dominant nonsyndromic hearing loss (DFNA56) [PMID:23936043].\",\n  \"teleology\": [\n    {\n      \"year\": 1991,\n      \"claim\": \"Establishing the complete modular architecture of human TNC—including 14.5 EGF-like repeats, 15 FNIII repeats, and alternatively spliced isoforms—provided the domain framework necessary for all subsequent receptor-mapping and signaling studies.\",\n      \"evidence\": \"cDNA sequencing with PCR-based splice variant identification and monoclonal antibody epitope mapping\",\n      \"pmids\": [\"1707164\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No functional assays for individual domains at this stage\", \"Stoichiometry of isoform expression across tissues not determined\"]\n    },\n    {\n      \"year\": 1992,\n      \"claim\": \"Solving the FNIII domain crystal structure at 1.8 Å revealed the immunoglobulin-superfamily fold and exposed RGD loop, explaining how TNC presents cell-adhesion motifs and providing the first atomic-level understanding of an ECM repeat module.\",\n      \"evidence\": \"X-ray crystallography with MAD phasing of selenomethionyl protein\",\n      \"pmids\": [\"1279805\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Only one of 15 FNIII repeats was structurally resolved\", \"No structures of EGF-like repeats or the fibrinogen-like globe\"]\n    },\n    {\n      \"year\": 1993,\n      \"claim\": \"Identification of multiple integrin receptors (αvβ3 via RGD, α2β1 via a non-RGD site) revealed that TNC engages the cell surface through mechanistically distinct, partially redundant adhesion pathways rather than a single integrin.\",\n      \"evidence\": \"Recombinant FNIII fragment adhesion assays, RGD mutagenesis, anti-integrin blocking antibodies, and affinity purification of integrins on TNC columns in endothelial cells\",\n      \"pmids\": [\"7694284\", \"7693733\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of α2β1 binding to TNC not defined\", \"Relative contribution of each integrin in vivo not determined\"]\n    },\n    {\n      \"year\": 1994,\n      \"claim\": \"Discovery that α9β1 binds TNC's third FNIII repeat at a non-RGD site, and that annexin II serves as a high-affinity receptor for the alternatively spliced FNIII segment, expanded the receptor repertoire beyond classical integrins and demonstrated isoform-specific signaling potential.\",\n      \"evidence\": \"α9 cDNA transfection with RGD mutagenesis (α9β1); radioligand Scatchard analysis, affinity chromatography, and anti-annexin II blocking (annexin II)\",\n      \"pmids\": [\"7523411\", \"7518469\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Downstream signaling from α9β1–TNC interaction not characterized\", \"In vivo relevance of annexin II–TNC interaction not tested\"]\n    },\n    {\n      \"year\": 1995,\n      \"claim\": \"Identification of α8β1 as an additional TNC integrin receptor reinforced the emerging picture of TNC as a multi-integrin ligand and showed that among RGD-containing ECM proteins, TNC has an unusually broad integrin-binding repertoire.\",\n      \"evidence\": \"α8 cDNA transfection in 293 cells, adhesion assays on recombinant TNC fragments, GRGDSP peptide elution from affinity columns\",\n      \"pmids\": [\"7559467\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional consequences of α8β1–TNC binding in tissue contexts undefined\"]\n    },\n    {\n      \"year\": 1997,\n      \"claim\": \"Demonstrating that TNC–αvβ3 interaction redistributes actin and clusters EGFR, increasing its tyrosine phosphorylation, established the first mechanistic link between TNC-mediated adhesion and growth factor receptor transactivation.\",\n      \"evidence\": \"Exogenous TNC on SMCs, β3-integrin cross-linking, EGFR phosphorylation assays, actin/EGFR colocalization by immunofluorescence\",\n      \"pmids\": [\"9314546\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether direct EGFR binding by TNC contributes was not tested\", \"In vivo relevance in vascular remodeling not shown\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Two discoveries redefined TNC's signaling capacity: its EGF-like repeats were shown to directly bind and activate EGFR (functioning as a matrix-tethered growth factor), and its binding to fibronectin's FNIII13 was shown to disrupt syndecan-4 coreceptor function, explaining TNC's anti-adhesive and pro-proliferative effects on the fibronectin matrix.\",\n      \"evidence\": \"EGFR autophosphorylation, ERK activation, and bead-tethered adhesion assays with EGF competition (EGFR); syndecan-4 rescue and FNIII13 competition on mixed FN/TNC substrata (syndecan-4)\",\n      \"pmids\": [\"11470832\", \"11731446\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Affinity of EGF-like repeats for EGFR much lower than EGF—physiological relevance of soluble presentation uncertain\", \"Whether syndecan-4 displacement occurs in vivo not demonstrated\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Placing TNC upstream of RhoA inactivation (via EGFR) and permissive for HGF/Rac-driven invasion established an epistatic pathway by which stromal TNC directs tumor cell morphological switching and invasive behavior.\",\n      \"evidence\": \"Collagen I/Matrigel invasion assays with dominant-negative/constitutively active Rho and Rac mutants, pharmacological inhibitors, myofibroblast co-culture\",\n      \"pmids\": [\"15059978\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo invasion assays not performed\", \"Whether TNC acts solely through EGFR-EGF-like repeats or also through integrins in this context is unresolved\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Identification of TNC as an endogenous TLR4 ligand—via its fibrinogen-like domain—fundamentally reframed TNC from a structural/adhesion molecule to a danger-associated molecular pattern (DAMP) that sustains chronic inflammation, as demonstrated by protection of Tnc-null mice from erosive arthritis and the role of periostin in incorporating TNC into ECM meshworks.\",\n      \"evidence\": \"Tnc-KO mice in arthritis model, TLR4-blocking antibodies and TLR4-deficient cells, human synovial cell stimulation (TLR4); periostin-null and TNC-null mouse phenocopying, domain-mapping binding assays (periostin)\",\n      \"pmids\": [\"19561617\", \"19887451\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Crystal structure of TNC fibrinogen-like domain bound to TLR4 not available\", \"Whether periostin-mediated TNC incorporation amplifies TLR4 signaling not directly tested\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Extension of the TLR4 mechanism to organ fibrosis (skin and lung) confirmed TNC as a general DAMP maintaining fibrosis amplification loops, while discovery of HIF1α-dependent transcriptional control downstream of IDH1 and tissue stiffness revealed a mechanosensitive feed-forward circuit in glioblastoma.\",\n      \"evidence\": \"TNC-KO mice in bleomycin skin/lung fibrosis models with fibroblast TLR4 signaling assays (fibrosis); IDH1 gain/loss-of-function xenografts with AFM stiffness measurements and HIF1α/miR-203 manipulation (glioblastoma)\",\n      \"pmids\": [\"27256716\", \"27820599\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether TLR4-TNC signaling is the sole driver or synergizes with integrin/EGFR pathways in fibrosis is unclear\", \"Quantitative threshold of ECM stiffness required to activate HIF1α-TNC loop not defined\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"miR-9-5p was validated as a direct post-transcriptional suppressor of TNC in chondrocytes, linking miRNA dysregulation to TNC-driven cartilage pathology in osteoarthritis.\",\n      \"evidence\": \"Luciferase reporter assay confirming miR-9-5p binding to Tnc 3'UTR, gain/loss-of-function in OA mouse model\",\n      \"pmids\": [\"31169312\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single study from one lab\", \"Whether miR-9-5p regulation of TNC operates in tissues beyond cartilage is unknown\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"miR-218 was confirmed as a second direct miRNA repressor of TNC, and TNC was placed within a TGFβ1/AKT/AP-1 positive feedback loop in glioma, further elucidating post-transcriptional control and downstream signaling.\",\n      \"evidence\": \"Dual-luciferase 3'UTR reporter, miR-218 overexpression/inhibition, nude mouse xenograft\",\n      \"pmids\": [\"34558654\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Relative importance of miR-218 vs miR-9-5p in controlling TNC levels in different tissues not compared\", \"Feedback loop not validated in non-glioma contexts\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"ChIP-based demonstration that Twist1 drives TNC expression indirectly through Prrx1 in fibroblasts provided the first two-step transcription factor cascade for TNC in kidney fibrosis, validated with fibroblast-specific Twist1 knockout mice.\",\n      \"evidence\": \"ChIP for Twist1 on Prrx1 promoter and Prrx1 on TNC promoter, fibroblast-specific Twist1-KO mice in UUO and IR injury\",\n      \"pmids\": [\"39181396\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether Twist1→Prrx1→TNC cascade operates in fibrosis of other organs not tested\", \"Direct versus Prrx1-mediated contribution of Twist1 not quantified\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Discovery that ER stress activates CREB5 via a super-enhancer to directly drive TNC transcription and EMT in hepatocellular carcinoma added a stress-responsive transcriptional input to the growing regulatory network.\",\n      \"evidence\": \"ChIP-seq for super-enhancer, ChIP for CREB5 on TNC promoter, CRISPR-Cas9 KO and overexpression\",\n      \"pmids\": [\"39915455\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single study; independent replication needed\", \"Whether CREB5-driven TNC activates TLR4 or EGFR in this context not examined\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key unresolved questions include: the structural basis of TNC fibrinogen-like domain recognition by TLR4; how multiple receptor systems (integrins, EGFR, TLR4, annexin II) are spatially and temporally coordinated on a single hexabrachion; and the in vivo hierarchy among transcriptional (HIF1α, Twist1/Prrx1, CREB5) and post-transcriptional (miR-218, miR-9-5p) regulatory inputs across different tissue and disease contexts.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No co-crystal structure of TNC-TLR4 or TNC-EGFR\", \"Relative signaling output from simultaneous engagement of multiple receptor types on a hexabrachion not quantified\", \"Integrated regulatory model across tissues lacking\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0098631\", \"supporting_discovery_ids\": [2, 3, 5, 6]},\n      {\"term_id\": \"GO:0048018\", \"supporting_discovery_ids\": [8, 11, 16]},\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": [0, 1, 12]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0031012\", \"supporting_discovery_ids\": [0, 1, 9, 12]},\n      {\"term_id\": \"GO:0005576\", \"supporting_discovery_ids\": [4, 11, 16]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1474244\", \"supporting_discovery_ids\": [0, 1, 9, 12]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [11, 16, 21]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [7, 8, 10, 17]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [10, 17, 22]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"EGFR\", \"ITGAV\", \"ITGB3\", \"ITGA2\", \"ITGB1\", \"ITGA9\", \"ANXA2\", \"POSTN\"],\n    \"other_free_text\": []\n  }\n}\n```"}